Fluorescence based techniques play an essential role in modern cell biology and biomedical research. Further development of these techniques has been hindered by the lack of suitable fluorescent probes. To overcome this challenge, various fluorescent nanoparticles have been developed. Among those, conjugated polymer dots (Pdots) exhibited superior properties such as high brightness, fast emission rate, and excellent photostability. However, a severe drawback associated with Pdots is their broad emission spectra, which significantly limit their usefulness in practical applications. This proposal describes the refinement of a new class of Pdots that emit at different wavelengths with narrow spectral bandwidth. To achieve this goal, we propose the following aims: (1) Develop multicolor bright polymer dots with emission bandwidth FWHM that is comparable to or less than 40 nm. Here, we will carry out various spectroscopic techniques to characterize the Pdot properties such as absorption cross section, emission bandwidth, fluorescence quantum yield, photostability, and fluorescence lifetime. Single-particle fluorescence imaging will also be performed to provide side-by-side brightness comparisons on Pdots versus inorganic quantum dots (Qdots) (2) Optimize nanoparticle surface properties to reduce nonspecific labeling. Here, to examine nonspecific labeling of Pdots, we will use a range of techniques, including gel electrophoresis, dynamic laser scattering, affinity chromatography, flow cytometry, and fluorescence spectroscopy/imaging. (3) Demonstrate multiplex detection of biomolecules in cellular environments. Here, we will use the bright, narrow-band Pdots for multiplex detection of biomolecules in cellular environments. Specifically, flow cytometry and fluorescence imaging will be performed to evaluate the labeling specificity and compare fluorescence brightness of the labeled targets. Reliable protocols will be established for simultaneous labeling of three or more cellular targets with multicolor Pdots. This will lay the foundation of applying these bright, narrow-band Pdots for multiplex detection and biological imaging.